Multipotent amniotic fetal stem cells

• US 7,569,385 B2
• Filed: 08/13/2004
• Issued: 08/04/2009
• Est. Priority Date: 08/14/2003
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Haas, Martin
• Primary Examiner(s): WEHBE, ANNE MARIE SABRINA
• Application Number:
  US10/918,739
• Publication Number:
  US 20050054093A1
• Time in Patent Office: 1,817 Days
• Field of Search: 435/325, 435/402, 435/455
• US Class Current: 435/325
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696

Abstract

A source of multipotent amniotic fluid/fetal stem cells (MAFSCs) is disclosed. MAFSC are of fetal origin and have a normal diploid karyotype. These cells are characterized by the following cell surface markers: SSEA3, SSEA4, Tra-1-60, Tra-1-81, Tra-2-54, HLA class I, CD13, CD44, CD49b, CD105 and are distinguished by the absence of the antigen markers CD34, CD45, and HLA Class II, but are distinguished from mouse embryonic stem cells in that these cells do not express the cell surface marker SSEA1. MAFSC express the stem cell transcription factor Oct-4. MAFSC cells can be propagated for an indefinite period of time in continuous culture in an undifferentiated state. The MAFSCs have the ability to differentiate in culture in a regulated manner, into three or more subphenotypes. Cells can then be differentiated into endodermal, mesodermal and ectodermal derived tissues in vitro and in vivo. A method for isolating, identifying, expanding and differentiating MAFSCs is disclosed.

Claims

1. A composition comprising isolated, mortal, epithelioid multipotent stem cells derived from amniotic fluid, wherein said stem cells are characterized by expression of SSEA3, SSEA4, Tra1-60, Tra1-81, Tra2-54, Oct-4 and CD105, and by less than 5% expression of SSEA1.

2. The composition of claim 1, wherein the stem cells are characterized by senescence after about 60 population doubling.

3. The composition of claim 1, wherein said cells are derived from a mammal.

4. The composition of claim 3, wherein said mammal is a human.

5. The composition of claim 1, wherein said stem cells are further characterized by expressing the following markers:

HLA class I, CD13, CD44, and CD49b.

6. A method of preparing multipotent stem cells, comprising:

harvesting amniotic fluid;

centrifuging the amniotic fluid;

plating cells onto plates coated with fibronectin in medium with 2% serum;

selecting colonies which adhere to the plates; and

isolating mortal, epithelioid stem cells.

7. The method of claim 6, further comprising allowing the population of stem cells to proliferate in 2% serum medium.

8. A method of preparing multipotent stem cells, comprising:

isolating cells from amniotic fluid;

culturing the cells on plates coated with fibronectin in 2% serum;

identifying and isolating mortal multipotent epithelioid stem cells based on appearance;

purifying the stem cells; and

growing the stem cells in or on a medium.

9. A method of preparing undifferentiated multipotent stem cells, comprising:

isolating cells from amniotic fluid;

culturing the cells on plates coated with fibronectin in 2% serum;

identifying and isolating mortal multipotent epithelioid stem cells based on appearance; and

expanding the cells in an undifferentiated state.

10. A genetically modified mortal epithelioid stem cell from amniotic fluid wherein said mortal epithelioid stem cell is characterized by expression of SSEA3, SSEA4, Tra1-60, Tra1-81, Tra2-54, Oct-4, and CD105, and by less than 5% expression of SSEA1.

11. The cell of claim 10, which has been genetically modified to express TERT.

12. A multipotent stem cell isolated from amniotic fluid, characterized by a) senescence in about 60 population doublings;

b) the presence of SSEA3, Tra1-60, Tra1-81, Tra2-54, Oct-4 and CD105; and

c) by less than 5% expression of SSEA1.